Conventional somatic cell nuclear transfer (SCNT) technique of in vitro production of cloned embryos involves use of costly and complicated micromanipulators. Handmade cloning (HMC) technique has been applied as efficient and cost-effective alternative in many livestock species. The aim of the present study was to compare the efficiency of in vitro production and in vitro development of cloned sheep embryos by the two techniques. Cloned embryos were produced by conventional SCNT using micromanipulator apparatus and by HMC technique. Enucleation efficiency and efficiency of fusion with somatic cell (nucleus donor) were compared. Cleavage percentage was observed on day 2 of in vitro culture (IVC), and morula and blastocyst percentages were calculated on day 7 of IVC. Higher enucleation efficiency (96.98 ± 1.01 vs. 93.62 ± 1.03; p > .05) as well as fusion efficiency was obtained with HMC technique than with conventional SCNT (96.26 ± 1.34 vs. 92.63 ± 0.70, p < .05); 181 cloned sheep embryos were produced in vitro by conventional SCNT and 92 by HMC. Cleavage percentage observed on day 2 of in vitro culture was higher in HMC than SCNT (66.92 ± 3.72 vs. 55.97 ± 2.5, respectively, p < .05). Morula percentage obtained was higher in SCNT than HMC (44.12 ± 2.93 vs. 30.43 ± 6.79, respectively, p < .05), whereas blastocyst percentage obtained by HMC was higher (12.46 ± 4.96) than SCNT (5.31 ± 2.25; p > .05). It was inferred that HMC technique provides a cost-effective and efficient method of in vitro production of cloned sheep embryos with a comparatively simpler technique with a possibility of automation. Efficiency of cloned embryo production could be improved further by propagating and standardizing this technique.

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Chlamydia abortus is a gram-negative obligate intracellular bacterium. Its lifecycle includes a resistant infectious form and a metabolically active non-infectious form. Chlamydia abortus infection results in abortion in goats; in nonpregnant animals the infection is usually subclinical. Chlamydia abortus presents a major zoonotic risk for pregnant women. The aim of this study was to investigate whether the embryonic zona pellucida (ZP) protects early embryo cells from infection and to test the efficacy of the washing protocol recommended by the IETS for bovine embryos. The study was performed in triple replicate: 14 donor goats, certified negative by ELISA and PCR to C. abortus, were synchronized, superovulated, and subsequently inseminated by males controlled negative for C. abortus. Fifty-two ZP-intact embryos (8–16 cells) were collected 4 days later, by laparotomy. The embryos were randomly divided into 12 batches. Nine batches of 5 embryos were incubated in a medium containing 4 × 107 Chlamydia mL–1, AB7 strain. After incubation for 18 h at 37°C in an atmosphere of 5% CO2, the embryos were washed in batches in 10 successive baths of PBS and 5% FCS solution in accordance with IETS guidelines for bovine embryos. In parallel, 3 batches of ZP-intact embryos (2, 2, and 3 embryos in the first, second, and third batches, respectively) were used as controls by being subjected to similar procedures, but without exposure to C. abortus. The 10 wash baths were collected separately and centrifuged for 1 h at 13 000 × g. The washed embryos and the pellets of the 10 centrifuged wash baths were frozen at –20°C before examination for evidence of C. abortus using RT-PCR. Chlamydia abortus DNA was found in all batches of infected ZP-intact embryos (9/9) after 10 successive washes. It was also detected in the tenth wash fluid for 4 batches (4/9) of infected embryos. As expected, none of the embryos or their washing fluids in the control batches were DNA positive. These results demonstrate that C. abortus adheres to and/or penetrates the ZP of in vivo caprine embryos after in vitro infection, and that the standard washing protocol recommended by the IETS for bovine embryos failed to remove it. The persistence of these bacteria after washing makes the embryo a potential means of transmission of the bacterium during embryo transfer from infected donor goat to healthy recipients and/or their offspring. Further studies are required to investigate whether enzymatic and/or antibiotic treatment of infected caprine embryos can eliminate C. abortus from the ZP.

Copro samples (1,126) of locally reared cattle at north Kashmir (Baramulla, Kupwara and Bandipora) were examined over a period of 1 year by qualitative and quantitative techniques. The overall prevalence of gastrointestinal (GI) helminths was 59.86% with trematodes, cestodes and nematodes in 11.63, 4.44 and 53.02% animals, respectively. Paramphistomes (7.10%) were the most prevalent trematodes followed by Fasciola spp. (4.53%) and Dicrocoelium spp. (0.27%). Prevalence of Moniezia spp. the only cestode found in the study was 4.44%. Among nematodes, the prevalence of strongyle worms was the highest (49.91%) followed by Trichuris spp. (3.64%) and Strongyloides spp. (2.04%). Seasonal variation revealed significantly highest prevalence of GI helminths during spring (68.21%) followed by autumn (67.50%), summer (55.59%) and winter (48.21%). Overall young cattle had significantly higher prevalence (68.13%) than adult animals (55.10%) but trematode infection was significantly higher in adults. Overall GI helminths were non-significantly higher in females (60.56%) as compared to males (58.24%) but the prevalence of paramphistomes was significantly higher in females. The prevalence of Dicrocoelium spp. and all the nematodes was nonsignificantly higher in males. EPG was ranging between 0 and 400 with an average EPG of 177.44±6.29. Highest EPG was recorded in spring (231.11±14.15) followed by summer (211.11±11.56), autumn (148.00±11.15) and winter (132.73±8.32). On coprocultural examination, Haemonchus spp. (65%) was found to be the most predominant strongyle worm followed by Bunostomum spp. (20%) and Trichostrongylus spp. (15%).